1. Field of the Invention
The present invention relates to a novel actin filament-binding protein "1-Afadin". More precisely, the present invention relates to a novel animal protein 1-Afadin that contributes to the cell-to-cell adherens junction (AJ) having an important role in individual formation of animals and pathigenesis.
2. Description of the Related Art
In various cellular events, such as cell adhesion, cell motility, and cell shape determination, specialized membrane domains are formed with transmembrane proteins, such as cell adhesion molecules, receptors, and channels, and these domains are often associated with the actin cytoskeleton (Biochem. Biophys, Acta 737:305-341, 1983; Curr. Opin. Cell Biol. 1:103-109, 1989; Cell Motil. Cytoskeleton 20:1-6, 1991; Curr. Opin. Cell Biol. 3:849-853, 1991; Science 258:955-964, 1992; Curr. Opin. Cell Biol. 4:834-839, 1992; Curr. Opin. Cell Biol. 5:653-660, 1993; Trends Biochem. Sci. 22:53-58, 1997). The linkage between the actin cytoskeleton and the plasma membrane plays a crucial role in these cellular events, and proteins linking the actin cytoskeleton to the transmembrane proteins have been identified. However, the molecular basis of the linkage between the actin cytoskeleton and the plasma membrane is not fully understood.
To understand this molecular linkage, the cell adhesion sites have been most extensively studied (Biochem. Biophys. Acta 737:305-341, 1983; Curr. Opin. Cell Biol. 1:103-109, 1989; Cell Motil. Cytoskeleton 20:1-6, 1991; Curr. Opin. Cell Biol. 3:849-853, 1991; Science 258:955-964, 1992; Curr. Opin. Cell Biol. 4:834-839, 1992; Curr. Opin. Cell Biol. 5:653-660, 1993; Trends Biochem. Sci. 22:53-58, 1997). As a result, the actin filament (F-actin)-associated cell adhesion sites are subclassed into two types: cell-to-cell and cell-to-matrix adherens junctions. Many linker proteins have been identified at the cell-to-cell AJ where cadherin interacts with each other at the extracellular surface (Development 102:639-655, 1988; Cell Motil. Cytoskeleton 20:1-6, 1991; Science 251:1451-1455, 1991; Curr. Opin. Cell Biol. 4:834-839, 1992; EMBO J. 8:1711-1717, 1989; Cell 65:849-857, 1991; Science 251:1451-1455, 1991; Curr. Opin. Cell Biol. 4:834-839, 1992). Among these binding proteins, .alpha.-catenin directly interacts with F-actin (Proc. Natl. Acad. Sci. U.S.A. 92, 8813-8817, 1995). .alpha.-Catenin also indirectly interacts with F-actin through .alpha.-actinin and/or ZO-1 (J. Cell. Biol. 130:67-77, 1995; J. Cell. Biol. 138:181-192, 1997). Further, Vinculin, another F-actin-binding protein, is concentrated at the cell-to-cell AJ, although its interacting molecules at cell-to-cell AJ have not yet been identified (Cell Motil. Cytoskeleton 20:1-6, 1991; Curr. Opin. Cell Biol. 4:834-839, 1992). At cell-to-matrix AJ where integrin interacts with matrix proteins at the extracellular surface, the cytoplasmic domain directly or indirectly interacts with F-actin-binding proteins, including .alpha.-actinin, vinculin, and talin (Ann. Rev. cell Dev. Biol. 11: 379-416, 1995).
As described above, many F-actin-binding proteins appear to serve as linkers of the actin cytoskeleton to the plasma membrane cadherin and integrin.
On the other hand, the linkage between the actin cytoskeleton and the plasma membrane is also important for neuron-specific events, such as growth cone pathfinding and subsequent formation and maintenance of synaptic junction (Neuron 1:761-772, 1988; Science 242:708-715, 1988; Curr. Opin. Neurobiol. 4:43-48, 1994; Curr. Opin. Neurobiol. 4:640-647, 1994; Cell 83:171-176, 1995). However, it remains to be clarified which molecules link the actin cytoskeleton to the plasma membrane in these neuron-specific events.
To address this issue, the inventors of the present patent application have isolated several novel F-actin-binding proteins from rat brain and analyzed the structure of proteins particularly specific to neural tissue and concentrated at synapse, from which an application for a patent has already been filed (Japanese Patent Application No. 9-92615). The protein of the prior invention (hereinafter, referred to as "neurabin" according to the inventor's name) has one F-actin-binding domain and one PDZ domain. The PDZ domain has been found in a variety of proteins, some of which are localized at cell-to-cell junctions, such as PSD-95/SAP90 at synaptic junction (Neuron 9:929-942, 1992; J. Biol. Chem. 268:4580-4583, 1993), Dlg at septate junction (Cell 66:451-464, 1991), ZO-1 and ZO-2 at tight junction (J. Cell Biol. 193:755-766, 1986; Proc. Natl. Acad. Sci. U.S.A. 88:3460-3464, 1991; J. Cell Biol. 121:491-502, 1993; J. Cell Biol. 123:1049-1053, 1993; Proc. Natl. Acad. Sci. U.S.A. 90:7834-7838, 1993; J. Cell Biol. 124:949-961, 1994). In addition, recent studies have revealed that the PDZ domain binds to unique C-terminal motifs of target proteins (Trends Biochem. Sci. 21:455-458, 1996), which are found in a large number of transmembrane proteins, such as N-methyl-D-aspartate receptor and Shaker-type K.sup.+ channel (Nature 378:85-88, 1995; Science 259:1737-1740, 1995; J. Neurosci. 16:2157-2163, 1996).
From the various findings described above, it is likely that neurabin, the protein of the prior invention found by the present inventors, serve as a linker of the actin cytoskeleton to a transmembrane protein(s) at synapse.
However, the whole molecular basis for the cell-to-cell adhesion has not yet been clarified, and it is necessary for such clarification to identify further actin filament-binding proteins. In addition, there is a possibility that these proteins leads to clarification of, for example, the mechanisms of infiltration and metastasis of carcinoma, and it is expected that these application allow for the development of diagnostic methods for malignancy of carcinoma, therapeutic methods thereof or agents for carcinoma and the like.
The present invention has been completed under such circumstance. An object of the present invention is to provide a novel actin filament-binding protein contributing to the cell-to-cell adhesion, and simultaneously clarifying its structure (amino acid sequence) and its properties.
Another object of the present invention is to provide a material for genetic engineering manipulation of the actin filament-binding protein.